Fenpyroximate Binds to the Interface between PSST and 49 kDa Subunits in Mitochondrial NADH-Ubiquinone Oxidoreductase.
Fenpyroximate Binds to the Interface between PSST and 49 kDa Subunits in Mitochondrial NADH-Ubiquinone Oxidoreductase.
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Fenpyroximate 与线粒体 NADH-泛醌氧化还原酶中 PSST 和 49 kDa 亚基之间的界面结合。
DOI：
10.1021/bi300047h
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Miyoshi H
中科院分区：
文献类型：
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作者：
Shiraishi Y;Murai M;Sakiyama N;Ifuku K;Miyoshi H
Nakamaru-Ogiso等人利用光亲和标记技术证明，牛心脏线粒体nadh -泛醌氧化还原酶（复合体I）的强抑制剂芬吡肟酸酯与ND5亚基结合[Nakamaru-Ogiso， E., et . (2003)Biochemistry 42,746 - 754]。然而，考虑到由跨膜螺旋1-15组成的ND5亚基的主体位于膜结构域的远端[Efremov， R. G., et al. (2010)Nature 465, 441-445]，他们的结果可能值得怀疑。由于确定膜结构域中抑制剂和/或醌结合位点的数量和位置对于阐明酶的功能是必要的，因此弄清除了亲水性和膜结构域之间的界面外是否还有其他抑制剂和/或醌结合位点是至关重要的。因此，我们使用两种新合成的芬邻肟酸衍生物[[125I]-4-叠氮苯基芬邻肟酸酯（[125I]APF）和[125I]-3-叠氮-5-碘苄基芬邻肟酸酯（[125I]AIF）]进行了光亲和标记实验，它们分别在药效核心部分和远离核心部分处具有光反应性叠氮基。双十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示[125I]APF和[125I]AIF分别与PSST和49 kDa亚基结合。对有限蛋白水解产生的标记的PSST和49 kDa亚基的断裂模式进行仔细检查表明，[125I]APF和[125I]AIF标记的残基分别位于Ser43-Arg66 （PSST）和Asp160-Arg174 （49 kDa）区域，这些区域面对PSST、49 kDa和ND1亚基界面形成的假定的醌结合袋。我们得出结论，芬吡肟酸酯不结合到膜结构域的远端，而是以一种方式驻留在两个结构域之间的界面上，使得抑制剂的药效吡唑环和侧链分别指向PSST和49 kDa亚基。这项研究回答了一个与复合体I有关的关键问题。
Using a photoaffinity labeling technique, Nakamaru-Ogiso et al. demonstrated that fenpyroximate, a strong inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I), binds to the ND5 subunit [Nakamaru-Ogiso, E., et al. (2003)Biochemistry 42, 746–754]. Considering that the main body of the ND5 subunit composed of transmembrane helixes 1–15 is located at the distal end of the membrane domain [Efremov, R. G., et al. (2010)Nature 465, 441–445], however, their result may be questionable. Because establishing the number and location of inhibitors and/or quinone binding sites in the membrane domain is necessary to elucidate the function of the enzyme, it is critical to clarify whether there is an additional inhibitor and/or quinone binding site besides the interface between the hydrophilic and membrane domains. We therefore performed photoaffinity labeling experiments using two newly synthesized fenpyroximate derivatives [[125I]-4-azidophenyl fenpyroximate ([125I]APF) and [125I]-3-azido-5-iodobenzyl fenpyroximate ([125I]AIF)] possessing a photoreactive azido group at and far from the pharmacophoric core moiety, respectively. Doubled sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that [125I]APF and [125I]AIF bind to the PSST and 49 kDa subunits, respectively. Careful examination of the fragmentation patterns of the labeled PSST and 49 kDa subunits generated by limited proteolysis indicated that the residues labeled by [125I]APF and [125I]AIF are located in the Ser43–Arg66 (PSST) and Asp160–Arg174 (49 kDa) regions, respectively, which face the supposed quinone-binding pocket formed at the interface of the PSST, 49 kDa, and ND1 subunits. We conclude that fenpyroximate does not bind to the distal end of the membrane domain but rather resides at the interface between the two domains in a manner such that the pharmacophoric pyrazole ring and side chain of the inhibitor orient toward the PSST and 49 kDa subunits, respectively. This study answers a critical question relating to complex I.